Anthrax is a highly infectious disease caused by Bacillus anthracis, and aerosolization of the dried bacterial spores is a major biological warfare and bioterrorism threat. Two plasmid-encoded anthrax toxins are essential for bacterial virulence. Edema toxin (EdTx) is comprised of edema factor (EF) and protective antigen (PA), while lethal toxin (LeTx) is a molecular complex of lethal factor (LF) and PA. EF is a secreted calmodulin-dependent adenylyl cyclase enzyme that causes tissue edema, and LF is a uniquely selective Zn++-metalloprotease that inactivates important cell-signaling enzymes (mitogen-activated protein kinase kinases [MAPKKs]) in mammalian cells. PA is the receptor-binding component, which delivers the catalytic components into the cytosol of cells. Our hypothesis is that novel drugs (specific inhibitors of anthrax toxins) can be prepared to reduce the virulence of these bacteria for humans/animals and provide a new therapeutic adjunct to antibiotic therapy and vaccination. The proposal is based on our extensive preliminary studies of new heterocyclic compounds (e.g., prostaglandin E2-L-histidine) that specifically block the adenylyl cyclase activity of EF, and knowledge of metalloprotease inhibitors that block LF activity. Objective 1 will evaluate the capacity o PGE2-L-histidine and PGE2-imidazole to reduce adenylyl cyclase activity of the EF toxin component using an in vitro enzyme assay. We will then use these data to design other inhibitors and dock them on the known crystal structures of EF and other adenylyl cyclases. Objective 2 will identify and characterize inhibitors that block the Zn++-metalloprotease activity of LF, and we will use these data in 3D-Quantitative structure activity relationship (QSAR) computations to optimize the enzyme inhibitors. Objective 3 will test the effectiveness of the EF and LF inhibitors in protecting cultured cells and mice challenged with toxins or B. anthracis. Objective 4 will evaluate the pharmacologic and toxicologic properties of these toxin inhibitors in experimental animals and establish their relative safety. Development of new drugs for anthrax by combining the inhibitors of EF and LF should reduce the virulence of B. anthracis, increase the efficacy of antibiotics, promote killing of the bacteria by phagocytes, and enhance vaccine-induced immunity.